EP3548888A1 - Dichtungsverstärker - Google Patents

Dichtungsverstärker

Info

Publication number
EP3548888A1
EP3548888A1 EP17818461.0A EP17818461A EP3548888A1 EP 3548888 A1 EP3548888 A1 EP 3548888A1 EP 17818461 A EP17818461 A EP 17818461A EP 3548888 A1 EP3548888 A1 EP 3548888A1
Authority
EP
European Patent Office
Prior art keywords
solution
patch clamp
ions
suitably
metal ions
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP17818461.0A
Other languages
English (en)
French (fr)
Other versions
EP3548888B1 (de
Inventor
Lars Damgaard LØJKNER
Anders Lindqvist
Daniel Rafael SAUTER
Kadla Røskva ROSHOLM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sophion Bioscience AS
Original Assignee
Sophion Bioscience AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sophion Bioscience AS filed Critical Sophion Bioscience AS
Priority to EP23155362.9A priority Critical patent/EP4209783A1/de
Priority claimed from PCT/EP2017/081388 external-priority patent/WO2018100206A1/en
Publication of EP3548888A1 publication Critical patent/EP3548888A1/de
Application granted granted Critical
Publication of EP3548888B1 publication Critical patent/EP3548888B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp

Definitions

  • a seal enhancer for improving the patch clamp seal in a patch clamp method or apparatus is provided.
  • a critical step in a planar patch clamp experiment is the seal formation i.e. formation of a tight bond between the cell membrane and the substrate of the planar patch clamp chip.
  • This seal poses a barrier for charge carriers such as ions and a good seal can therefore be measured as a high seal resistance.
  • the exact mechanism by which this connection occurs remains to be completely understood, however, it is known that small gaps between the cell and the substrate can be closed (sealed) by precipitation of a salt. To exploit this effect, it is important to have both components of this salt (i.e. anions and cations) separated in internal and external solution; otherwise, the salt would already start to precipitate before the cell is applied and this would result in an altered ionic strength of the solution.
  • Solubility of a solution is defined in the solubility product K sp .
  • K sp the solubility product
  • the smaller K sp the fewer ions are freely available and the more are present as solid salt.
  • R me m b r a n e was historically enhanced using fluoride in the internal solution and Ca 2+ in the external.
  • CaF 2 has a K sp of 3.58 x 10 "11 (McCann, 1968), thus CaF 2 precipitates at the interface between internal and external solution thus fostering seal formation (Vargas, Yeh, Blumenthal, & Lucero, 1999).
  • Ca 2+ in the internal solution and in the external solution e.g. Ca 2+ -activated K + and CI " channels
  • the use of F- in these solutions is not feasible due to the low K sp .
  • the concentration of metal ions in the external solution is in the region of 50- lOOmM, cf. EP1775586.
  • too metal ion concentrations may possibly affect ion channel function in an adverse manner leading to incorrect responses and inaccuracies in drug target responses.
  • a patch clamp system comprising a patch clamp device, an internal solution and an external solution.
  • the internal solution comprises one or more anions selected from phosphate (P0 4 3 ⁇ ) ions, sulfate (S0 4 2 ⁇ ) ions and fluoride (F ) ions
  • the external solution comprises one or more metal ions selected from Ca 2+ , Sr 2+ and Ba 2+ , or combinations thereof.
  • the concentration of said metal ions in the external solution is between 1 and 20mmol/L.
  • anions selected from phosphate (P0 4 3 ⁇ ), sulfate (S0 4 2 ⁇ ) and fluoride (F ) anions in combination with one or more metal ions selected from Ca 2+ , Sr 2+ and Ba 2+ , or combinations thereof as a seal enhancer in a patch clamp system is also provided, wherein the anions are present in an internal solution, and the metal ions are present in an external solution of the patch clamp system.
  • the concentration of said metal ions in the external solution is between 1 and 20mmol/L
  • a method for providing a gigaseal in a patch clamp system in which the patch clamp system comprises a patch clamp device, an internal solution and an external solution.
  • the method comprising the steps of: a introducing a solution of one or more anions selected from phosphate (P0 4 3 ⁇ ) sulfate (S0 4 2 ⁇ ) and fluoride (F ) anions into the internal solution; b introducing a solution of cells into the external solution, a nd capturing the cells within the patch clamp device; c introducing a solution of one or more metal ions selected from Ca 2+ , Sr 2+ and
  • steps a and b. can be performed in any order.
  • Fig. 1 shows a simplified view of a patch clamp device 102 for use in patch clamp analysis of a cell 120 sealed to the chip 102 to form a gigaseal.
  • the device 102 separates an external solution 122 on a first side of the device from an internal solution 124 on an opposite side thereof.
  • the diameter a of the cell is approximately 5-20 ⁇ , such as approximately 10 ⁇ .
  • a hole 108 is formed in the device 102, through which suction may be applied to secure the cell 120 in place over the hole 108.
  • Electrodes (not shown) may be provided in both sides of the device in order to determine an electrical resistance, a flow of ions or a voltage difference across the cell and through the hole 108.
  • external solution refers to "extracellular physiological solution", external meaning outside the cell.
  • intermediate solution refers to "intracellular physiological solution", internal meaning inside the cell or the solution intended to provide electrical and fluidic contact with the inside of the cell.
  • a patch clamp system is provided, as illustrated schematically in Figure 1.
  • the patch clamp system comprises a patch clamp device 102, an internal solution 124 and an external solution 122.
  • the patch clamp system may be useful for determining or monitoring current flow through ion channel-containing structures such as cell membranes, with a high throughput and reliability and under conditions that are realistic with respect to the influences to which the cells or cell membranes are subjected.
  • the results determined e.g., variations in ion channel activity as a result of influencing the cell membrane with, e.g., various test compounds, can be relied upon as true manifestations of the proper influences and not of artefacts introduced by the measuring system, and can be used as a valid basis for studying electrophysiological phenomena related to the conductivity or capacitance of cell membranes under given conditions.
  • the current through one or more ion channels is directly measured using reversible electrodes as characterized below, typically silver/silver halide electrodes such as silver chloride electrodes, as both measuring electrodes and reference electrodes.
  • the patch clamp device may comprise an array of a plurality of chips on a single carrier. It may be is used in a method for determining and/or monitoring electrophysiological properties of ion channels in ion channel-containing structures, typically lipid membrane-containing structures such as cells, by establishing an electrophysiological measuring configuration in which a cell membrane forms a high resistive seal around a measuring electrode, making it possible to determine and monitor a current flow through the cell membrane.
  • the patch clamp system is for example useful in a method for analysing the electrophysiological properties of a cell membrane comprising a glycocalyx.
  • the patch clamp system may be used in or form part of an apparatus for studying electrical events in cell membranes, such as an apparatus for carrying out patch clamp techniques utilised to study ion transfer channels in biological membranes.
  • the internal solution comprises one or more anions selected from phosphate (P0 4 3 ⁇ ), sulfate (S0 4 2 ⁇ ) and fluoride (F ) anions, preferably sulfate (S0 4 2 ⁇ ) ions.
  • concentration of anions, such as sulfate (S0 4 2 ⁇ ) ions, in the internal solution is between 20 and 200mmol/L, suitably between 45 and 175mmol/L, more suitably between 50 and 150mmol/L, even more suitably between 75 and 125mmol/L, inclusive, most suitably around lOOmmol/L.
  • S0 4 2 does not inhibit any ion channel or relevant up-stream pathway.
  • sulfate salts are highly soluble in water, under physiological conditions. This allows a relatively broad spectrum of anions to be incorporated in the internal solution.
  • sulfate ions are provided in the internal solution in the form of Mg 2+ , Ca 2+ , K + or Na + salts, preferably K + or Na + salts.
  • the internal solution is suitably substantially free from ions which may cause sulfate precipitation.
  • the internal solution is preferably substantially free from Ba 2+ , Fe 3+ or Be 2 ions, and is preferably completely free from Ba 2+ , Fe 3+ or Be 2 ions.
  • the external solution comprises one or more metal ions selected from Ca 2+ , Sr 2+ and Ba 2+ , or combinations thereof.
  • the metal ions are Ba 2+ .
  • the concentration of the metal ions in the external solution is between 1 and 20mmol/L, suitably between 4 and 17mmol/L, more suitably between 5 and 15mmol/L, even more suitably between 7.5 and 12.5mmol/L, inclusive, most suitably around lOmmol/L.
  • Ba 2+ is known to inhibit some K + channels in the ⁇ to low mM - range (Wulff & Zhorov, 2008) . However, to the best of our knowledge, Ba 2+ does not have any effect on any of the CI " channels.
  • the here described seal enhancer will also be useful for patch clamp applications where Ba 2+ functions as an inhibitor (e.g. inward rectifier K + channels).
  • the external solution is suitably substantially free of ions which may cause metal ion precipitation.
  • the external solution is preferably substantially free from sulfate, and is preferably completely free from sulfate ions.
  • the one or more metal ions are suitably added in the form of their CI " salt.
  • all cations in the external solution are CI " .
  • the internal solution may have a concentration of fluoride (F ) ions which is less than 5mmol/L, suitably less than lmmol/L, and is more preferably zero. Fluoride ions can therefore be reduced and/or eliminated.
  • F fluoride
  • the use of one or more anions selected from phosphate (P0 4 3 ), sulfate (S0 4 2 ) and fluoride (F ) anions in combination with one or more metal ions selected from Ca 2+ , Sr 2+ and Ba 2+ , or combinations thereof as a seal enhancer in a patch clamp system is provided.
  • the use of sulfate (S0 4 2 ) ions in combination with metal ion selected from Ba 2+ as a seal enhancer in a patch clamp system is provided.
  • the anions are present in an internal solution, and the metal ions are present in an external solution of the patch clamp system.
  • the concentration of said metal ions in the external solution is between 1 and 20mmol/L.
  • a method for providing a gigaseal in a patch clamp system comprises a patch clamp device, an internal solution and an external solution.
  • the method comprises the steps of: a. introducing a solution of one or more anions selected from phosphate (P0 4 3 ), sulfate (S0 4 2 ) and fluoride (F ) anions into the internal solution; b. introducing a solution of cells into the external solution, and capturing the cells within the patch clamp device; c.
  • the method comprises the steps of: a. introducing a solution of sulfate (S0 4 2 ⁇ ) ions into the internal solution;
  • concentration of the Ba 2+ metal ions in the external solution is between 1 and 20mmol/L.
  • steps a and b. can be performed in any order; i.e. a solution of cells can be introduced to the external solution, and captured within the patch clamp device before sulfate solution is introduced in the external solution. However, it is preferred that step a. is performed before step b.
  • the method may further comprise the step of (d) performing patch clamp measurements on the cell(s) in the patch clamp device.
  • the concentration of anions, in particular sulfate (S0 4 2 ⁇ ) anions, in the internal solution is between 20 and 200mmol/L, suitably between 45 and 175mmol/L, more suitably between 50 and 150mmol/L, inclusive.
  • the metal ions are suitably Ba 2+ .
  • the concentration of the metal ions in the external solution is suitably between 4 and 17mmol/L, more suitably between 5 and 15mmol/L, inclusive.
  • Cells for use in the method may be selected from CHO or HEK293.
  • S0 4 2 ⁇ has a relatively high solubility with Ca 2+ .
  • the solubility constant (Ksp) of CaS0 4 is 4.93xl0 "5 .
  • the present technology is useful for applications where Ca 2+ should have a precise internal concentration over the entire course of the experiment.
  • the internal solution and/or the external solution may additionally comprise calcium (Ca 2+ ) ions.
  • EXAMPLE 1 Test of ion pairs as seal enhancers in Qube experiment
  • Cell line TE671 expressing the Na v 1.4 Channel Tested ion pairs: CaF 2 (reference), SrF 2 , BaS0 4 , Ca 3 (P0 4 ) 2
  • the cation in the extracellular (EC) solutions were applied at low concentration (2 mM) during cell addition and positioning and at high concentration (10 mM) before sealing and current measurement.
  • the osmolality of the internal solutions is adjusted to 350mOsm with KCI .
  • the pH of the internal solutions is adjusted to pH 7.0 using KOH. Test procedure
  • HEK293 cells stably expressing the hTMEM16A ion channel General method: The internal solutions (at various concentrations of sulfate) are provided to the internal side of the Qube device. A priming solution, containing cells as specified, is then provided to the external side of the Qube device, and cells are captured. The external solutions (at various Ba 2+ concentrations) are then provided to the external side of the device.
  • Table 3 Summary of R m embrane from 5 individual experiments (QChips) measured on CHO- hERG cells. Statistical significance was assessed using an ANOVA test. * depicts significance with respect to the next lower concentration of Barium and # refers to significance to the next lower sulfate concentration. * or # indicate p ⁇ 0.05; ** or ## indicate p ⁇ 0.01; *** or ### indicate p ⁇ 0.001. *
  • Table 4 Summary of Rmembrane from 1 experiment (QChips) measured on HEK-hTMEM16A cells. Statistical significance was assessed using an ANOVA test. * depicts significance with respect to the next lower concentration of Barium and # refers to significance to the next lower sulfate concentration. * or # indicate p ⁇ 0.05; ** or ## indicate p ⁇ 0.01 ; *** or ### indicate p ⁇ 0.001. *

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
EP17818461.0A 2016-12-02 2017-12-04 Dichtungsverstärker Active EP3548888B1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP23155362.9A EP4209783A1 (de) 2016-12-02 2017-12-04 Dichtungsverstärker

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DKPA201670957 2016-12-02
US201762449278P 2017-01-23 2017-01-23
PCT/EP2017/081388 WO2018100206A1 (en) 2016-12-02 2017-12-04 Seal enhancer

Related Child Applications (2)

Application Number Title Priority Date Filing Date
EP23155362.9A Division EP4209783A1 (de) 2016-12-02 2017-12-04 Dichtungsverstärker
EP23155362.9A Division-Into EP4209783A1 (de) 2016-12-02 2017-12-04 Dichtungsverstärker

Publications (2)

Publication Number Publication Date
EP3548888A1 true EP3548888A1 (de) 2019-10-09
EP3548888B1 EP3548888B1 (de) 2023-04-05

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ID=60788557

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EP23155362.9A Withdrawn EP4209783A1 (de) 2016-12-02 2017-12-04 Dichtungsverstärker
EP17818461.0A Active EP3548888B1 (de) 2016-12-02 2017-12-04 Dichtungsverstärker

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Application Number Title Priority Date Filing Date
EP23155362.9A Withdrawn EP4209783A1 (de) 2016-12-02 2017-12-04 Dichtungsverstärker

Country Status (4)

Country Link
US (2) US11215606B2 (de)
EP (2) EP4209783A1 (de)
JP (1) JP7086072B2 (de)
DE (1) DE202017007491U1 (de)

Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2788695A (en) * 1994-10-28 1996-05-23 Neurosearch A/S Patch clamp apparatus and technique having high throughput and low fluid volume requirements
WO1999005515A1 (en) * 1997-07-24 1999-02-04 Orion Research, Inc. Low detection limit ion selective membrane electrodes
WO1999028342A2 (en) 1997-12-03 1999-06-10 Merck & Co., Inc. Low-voltage activated calcium channel compositions and methods
US20040168912A1 (en) * 2000-02-11 2004-09-02 James Klemic Planar patch clamp electrodes
CA2428698C (en) 2000-11-13 2012-08-21 The Queen's Medical Center Methods of screening for ltrpc7 modulators
US6649669B2 (en) * 2001-12-28 2003-11-18 American Dental Association Health Foundation Single solution bonding formulation
WO2003080570A2 (en) 2002-03-20 2003-10-02 Transmolecular, Inc. Recombinant expression vectors for functional nav1.9 sodium channels
EP1775586B1 (de) 2005-10-17 2011-12-14 Nanion Technologies GmbH Verfahren zur elektrophysiologischen Untersuchung einer einen Ionenkanal umfassenden Membran
JP2010532748A (ja) * 2007-04-26 2010-10-14 メルク・シャープ・エンド・ドーム・コーポレイション カルシウムチャネル遮断薬としての２−置換インドール誘導体
CA2695216A1 (en) * 2007-07-31 2009-02-05 Purdue Pharma L.P. Dielectric spectroscopy assays for screening of ion channel ligands
FR2921935B1 (fr) * 2007-10-05 2011-10-28 Centre Nat Rech Scient Procede de criblage de composes utilisables pour le traitement de troubles respiratoires
GB0812192D0 (en) 2008-07-03 2008-08-13 Lectus Therapeutics Ltd Calcium ion channel modulators & uses thereof
US9488637B2 (en) * 2011-01-26 2016-11-08 The Curators Of The University Of Missouri Combination of single-cell electroporation and electrical recording using the same electrode
US9150598B2 (en) * 2011-10-05 2015-10-06 The Regents Of The University Of California Masking apertures enabling automation and solution exchange in sessile bilayers
JP6037717B2 (ja) * 2011-12-20 2016-12-07 国立研究開発法人科学技術振興機構 プレーナーパッチクランプ装置、該装置用電極部及び細胞イオンチャンネル電流計測方法
KR102141898B1 (ko) 2012-12-18 2020-08-06 바이오크린 에이비 당뇨병의 치료 및/또는 진행의 제한 방법
HUE045351T2 (hu) 2013-05-17 2019-12-30 Univ New York State Res Found Befelé egyenirányító áram elektronikus expressziója humán indukált pluripotens õssejtekbõl származó szívizomsejtekben
US9977009B2 (en) * 2013-08-30 2018-05-22 Japan Science And Technology Agency Planar patch clamp device and planar patch clamp system
WO2017152064A1 (en) * 2016-03-03 2017-09-08 Ceramatec, Inc. Intercalation membrane

Also Published As

Publication number Publication date
US11692994B2 (en) 2023-07-04
JP2020513242A (ja) 2020-05-14
US11215606B2 (en) 2022-01-04
JP7086072B2 (ja) 2022-06-17
US20220107304A1 (en) 2022-04-07
EP4209783A1 (de) 2023-07-12
US20190383792A1 (en) 2019-12-19
DE202017007491U1 (de) 2022-01-07
EP3548888B1 (de) 2023-04-05

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